Landmann F et al. (2012) Parasit Vectors "Efficient in vitro RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, Brugia malayi."

Foster JM, Landmann F, Slatko BE, Sullivan W

[Parasit Vectors, 2012]

BACKGROUND: RNA interference (RNAi) is an efficient reverse genetics technique for investigating gene function in eukaryotes. The method has been widely used in model organisms, such as the free-living nematode Caenorhabditis elegans, where it has been deployed in genome-wide high throughput screens to identify genes involved in many cellular and developmental processes. However, RNAi techniques have not translated efficiently to animal parasitic nematodes that afflict humans, livestock and companion animals across the globe, creating a dependency on data tentatively inferred from C. elegans. RESULTS: We report improved and effective in vitro RNAi procedures we have developed using heterogeneous short interfering RNA (hsiRNA) mixtures that when coupled with optimized immunostaining techniques yield detailed analysis of cytological defects in the human parasitic nematode, Brugia malayi. The cellular disorganization observed in B. malayi embryos following RNAi targeting the genes encoding -tubulin, and the polarity determinant protein, PAR-1, faithfully phenocopy the known defects associated with gene silencing of their C. elegans orthologs. Targeting the B. malayi cell junction protein, AJM-1 gave a similar but more severe phenotype than that observed in C. elegans. Cellular phenotypes induced by our in vitro RNAi procedure can be observed by immunofluorescence in as little as one week. CONCLUSIONS: We observed cytological defects following RNAi targeting all seven B. malayi transcripts tested and the phenotypes mirror those documented for orthologous genes in the model organism C. elegans. This highlights the reliability, effectiveness and specificity of our RNAi and immunostaining procedures. We anticipate that these techniques will be widely applicable to other important animal parasitic nematodes, which have hitherto been mostly refractory to such genetic analysis.

Landmann F et al. (2004) Dev Biol "Multiple regulatory elements with spatially and temporally distinct activities control the expression of the epithelial differentiation gene lin-26 in C. elegans."

Quintin S, Labouesse M, Landmann F

[Dev Biol, 2004]

Epithelial differentiation is a very early event during development of most species. The nematode Caenorhabditis elegans, with its well-defined and invariant lineage, offers the possibility to link cell lineage, cell fate specification and gene regulation during epithelial differentiation. Here, we focus on the regulation of the gene lin-26, which is required for proper differentiation of epithelial cells in the ectoderm and mesoderm (somatic gonad). lin-26 expression starts in early embryos and remains on throughout development, in many cell types originating from different sublineages. Using GFP reporters and mutant rescue assays, we performed a molecular dissection of the lin-26 promoter and could identify almost all elements required to establish its complex spatial and temporal expression. Most of these elements act redundantly, or synergistically once combined, to drive expression in cells related by function. We also show that lin-26 promoter elements mediate activation in the epidermis (hypodermis) by the GATA factor ELT-1, or repression in the foregut (pharynx) by the FoxA protein PHA-4. Taken together, our data indicate that lin-26 regulation is achieved to a large extent through

Landmann F et al. (2010) PLoS Negl Trop Dis "Asymmetric Wolbachia segregation during early Brugia malayi embryogenesis determines its distribution in adult host tissues."

Slatko B, Sullivan W, Landmann F, Foster JM

[PLoS Negl Trop Dis, 2010]

Wolbachia are required for filarial nematode survival and fertility and contribute to the immune responses associated with human filarial diseases. Here we developed whole-mount immunofluorescence techniques to characterize Wolbachia somatic and germline transmission patterns and tissue distribution in Brugia malayi, a nematode responsible for lymphatic filariasis. In the initial embryonic divisions, Wolbachia segregate asymmetrically such that they occupy only a small subset of cells in the developing embryo, facilitating their concentration in the adult hypodermal chords and female germline. Wolbachia are not found in male reproductive tissues and the absence of Wolbachia from embryonic germline precursors in half of the embryos indicates Wolbachia loss from the male germline may occur in early embryogenesis. Wolbachia rely on fusion of hypodermal cells to populate adult chords. Finally, we detect Wolbachia in the secretory canal lumen suggesting living worms may release bacteria and/or their products into their host.

Landmann F et al. (2014) PLoS Negl Trop Dis "Co-evolution between an endosymbiont and its nematode host: Wolbachia asymmetric posterior localization and AP polarity establishment."

Foster JM, Slatko BE, Michalski ML, Sullivan W, Landmann F

[PLoS Negl Trop Dis, 2014]

While bacterial symbionts influence a variety of host cellular responses throughout development, there are no documented instances in which symbionts influence early embryogenesis. Here we demonstrate that Wolbachia, an obligate endosymbiont of the parasitic filarial nematodes, is required for proper anterior-posterior polarity establishment in the filarial nematode B. malayi. Characterization of pre- and post-fertilization events in B. malayi reveals that, unlike C. elegans, the centrosomes are maternally derived and produce a cortical-based microtubule organizing center prior to fertilization. We establish that Wolbachia rely on these cortical microtubules and dynein to concentrate at the posterior cortex. Wolbachia also rely on PAR-1 and PAR-3 polarity cues for normal concentration at the posterior cortex. Finally, we demonstrate that Wolbachia depletion results in distinct anterior-posterior polarity defects. These results provide a striking example of endosymbiont-host co-evolution operating on the core initial developmental event of axis determination.

Vuong, Thanh TK et al. (2013) International Worm Meeting "Mechanical forces in C. elegans embryo elongation."

Labouesse, Michel, Vuong, Thanh TK

[International Worm Meeting, 2013]

Embryo development requires precise coordination of mechanical forces and their failure can lead to diseases. During the morphogenesis of C. elegans embryo, the cooperation of epidermal acto-myosin network and muscle contractions is essential. The acto-myosin activity in the epidermis, which has been shown to be more important in lateral than in dorsal-ventral cells, squeeze the embryo and make it elongate [1]. Muscle contractions become active around 1.7-1.8 fold stage. They have been showed to induce a mechano-transduction pathway [2], which is important for elongation. However, it is unclear how the contractions along the anterior-posterior axis help to increase the length of the embryo. Our project aims to elucidate the mechanical role of muscle contractions and its coordination with acto-myosin forces. The experiments are designed following a working model where muscle contractions induce a change in the elasticity of the embryo. We are using a laser nano-dissection technique to investigate cortical tension and elasticity of epidermal cells before and after the onset of muscle contractions. In parallel, we are evaluating the relative changes of acto-myosin forces with a FRET sensor [3] inserted in HMP-1 - a component of the adherens junctions. I will present our observations and preliminary results of the epidermal cortex nano-dissection experiments and measures of acto-myosin forces exerted on adherens junctions. References 1.Gally C, Wissler F, Zahreddine H, Quintin S, Landmann F, Labouesse M. Myosin II regulation during C. elegans embryonic elongation: LET-502/ROCK, MRCK-1 and PAK-1, three kinases with different roles. Development. 2009 Sep;136(18):3109-19. Epub 2009 Aug 12. 2.Zhang H, Landmann F, Zahreddine H, Rodriguez D, Koch M, Labouesse M. A tension-induced mechanotransduction pathway promotes epithelial morphogenesis. Nature. 2011 Mar 3;471(7336):99-103. 3.Grashoff C, Hoffman B, Brenner M, Zhou R, Parsons M, Yang M, McLean M, Sligar S, Chen C, Ha T, Schwartz M. Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature. 2010 July 8; 466(7303): 263-266.

Kipreos ET et al. (2000) Genome Biol "The F-box protein family."

Kipreos ET, Pagano M

[Genome Biol, 2000]

SUMMARY: The F-box is a protein motif of approximately 50 amino acids that functions as a site of protein-protein interaction. F-box proteins were first characterized as components of SCF ubiquitin-ligase complexes (named after their main components, Skp I, Cullin, and an F-box protein), in which they bind substrates for ubiquitin-mediated proteolysis. The F-box motif links the F-box protein to other components of the SCF complex by binding the core SCF component Skp I. F-box proteins have more recently been discovered to function in non-SCF protein complexes in a variety of cellular functions. There are 11 F-box proteins in budding yeast, 326 predicted in Caenorhabditis elegans, 22 in Drosophila, and at least 38 in humans. F-box proteins often include additional carboxy-terminal motifs capable of protein-protein interaction; the most common secondary motifs in yeast and human F-box proteins are WD repeats and leucine-rich repeats, both of which have been found to bind phosphorylated substrates to the SCF complex. The majority of F-box proteins have other associated motifs, and the functions of most of these proteins have not yet been defined.

Chamberlin HM et al. (1995) Worm Breeder's Gazette "lin-49, An Essential Gene Required For Normal F and U Cells."

Chamberlin HM, Sternberg PW, Baillie DL

[Worm Breeder's Gazette, 1995]

lin-49, an essential gene required for normal F and U cells

Bennett JL et al. (1988) Parasitol Today "Pharmacology of ivermectin."

Williams JF, Bennett JL, Dave V

[Parasitol Today, 1988]

Ivermectin is a semi-synthetic macrocyclic lactone (Fig. I) active in single low doses against many parasites - particularly nematodes and arthropods. It has been registered for animal health use since early 1985, and was earlier this year approved for human use by the French Directorate o f Pharmacy and Drugs. Of particular interest is ivermectin's potential as a micro filaricide for treatment o f onchocerciasis. Clinical trials leave little doubt about the potential o f ivermectin as a therapeutic tool for symptomatic relief from the effects o f infection with Onchocerca volvulus, and the drug is also recognized to have potential in reducing transmission o f the parasite. The manufacturers (Merck, Sharp and Dohme) recently arranged to provide the drug free o f charge to the WHO for mass trials against onchocerciasis in 12 African and Central American countries. In this article we focus on the pharmacological properties o f ivermectin, with a brief consideration of its absorption, fate, excretion and side-effects, and a discussion o f its micro filaricidal action.

Rehain K et al. (2015) Curr Biol "Neuron migration: anillin protects leading edge actin."

Maddox AS, Rehain K

[Curr Biol, 2015]

Establishment of a neuronal system requires proper regulation of the F-actin-rich leading edges of migrating neurons and neurite growth cones. A new study shows that RhoG signals through the multi-domain protein anillin to stabilize F-actin in these structures.

Kwiatkowski AV et al. (2010) Proc Natl Acad Sci U S A "In vitro and in vivo reconstitution of the cadherin-catenin-actin complex from Caenorhabditis elegans."

Hardin J, Weis WI, Nelson WJ, Choi HJ, Lynch AM, Kwiatkowski AV, Benjamin JM, Pokutta S, Maiden SL

[Proc Natl Acad Sci U S A, 2010]

The ternary complex of cadherin, beta-catenin, and alpha-catenin regulates actin-dependent cell-cell adhesion. alpha-Catenin can bind beta-catenin and F-actin, but in mammals alpha-catenin either binds beta-catenin as a monomer or F-actin as a homodimer. It is not known if this conformational regulation of alpha-catenin is evolutionarily conserved. The Caenorhabditis elegans alpha-catenin homolog HMP-1 is essential for actin-dependent epidermal enclosure and embryo elongation. Here we show that HMP-1 is a monomer with a functional C-terminal F-actin binding domain. However, neither full-length HMP-1 nor a ternary complex of HMP-1-HMP-2(beta-catenin)-HMR-1(cadherin) bind F-actin in vitro, suggesting that HMP-1 is auto-inhibited. Truncation of either the F-actin or HMP-2 binding domain of HMP-1 disrupts C. elegans development, indicating that HMP-1 must be able to bind F-actin and HMP-2 to function in vivo. Our study defines evolutionarily conserved properties of alpha-catenin and suggests that multiple mechanisms regulate alpha-catenin binding to F-actin.